A method for analysis of reducing sugars is known by subjecting a sample to liquid chromatography using water as the mobile phase, adding to the eluate thus obtained an aqueous solution of boric acid containing a basic amino acid such as arginine, allowing a reaction to proceed under heating, then cooling the reaction mixture solution and irradiate it with excitation light and measuring the intensity of fluorescence or absorbance (Patent Document 1). The apparatus used there is made up of a liquid chromatograph with an extended flow path, to which is connected a supply channel for a boric acid solution containing a basic amino acid, and to which flow path are further attached a heating device, a cooling device, an excitation light irradiator, and a device measuring fluorescence intensity. According to this method, a supply channel is indispensable to add a boric acid solution containing a basic amino acid to the eluate from the chromatograph to allow a reaction to take place.
Further, as an improved type of the above method, a method is known in which a sample is applied to liquid chromatography using a mobile phase containing a basic amino acid such as arginine and boric acid, which are the reagents for the reaction, and is eluted, and the eluate thus obtained is heated to let the reagents and saccharides react (heat reaction) and then cooled, and this reaction mixture solution then is measured for intensity of fluorescence emitted under irradiation with excitation light, or for absorbance (Patent Document 2). In this method, as an aqueous solution of boric acid containing a basic amino acid is employed as the mobile phase for liquid chromatography, there is no need for providing a supply channel for addition of an aqueous solution of boric acid containing a basic amino acid in order for allowing a reaction to proceed. The above methods for analysis of reducing sugars are called post-column fluorometric determination-boric acid complex anion exchange method.
The methods for analysis of reducing sugars mentioned above both utilize in detection the production of a strong fluorogenic derivative through the heat reaction of reducing sugars with a basic amino acid, such as arginine, in the presence of boric acid (Non-patent Document 1). This strong fluorogenic derivatives is brown-colored melanoidine formed by the heat reaction (Maillard reaction) of reducing sugars and a basic amino acid, i.e., an amino compound, and it emits light at the wavelength of 430 nm when irradiated with excitation light at the wavelength of 320 nm. These method for analysis of reducing sugars utilize that reducing sugars have a property of readily binding to boric acid to form an anionic complex ion, and that this anionic complex ion is retained in anion exchanger column chromatography.
In the post-column fluorometric determination-boric acid complex anion exchange method, an aqueous solution (pH 7-10) is employed which contains a basic amino acid at a concentration of 0.01-5% and boric acid at a concentration of 0.05-0.5 M. Those amino acids which are employed there are arginine, lysine, histidine, and the like. Recently, it is further known to perform analysis of sugars employing as a mobile phase for liquid chromatography a gradient formed between a 0.1 M borate buffer and a 0.4 M borate buffer (Patent Documents 3, 4).
In the post-column fluorometric determination-boric acid complex anion exchange method, elution of a sample by liquid chromatography is carried out at temperatures of from room temperature to 70° C., and the heat reaction (Maillard reaction) is let proceed at 140-180° C. Since elution and reaction are performed at such high temperatures and at high boric acid concentrations, boric acid contained in the mobile phase sometimes precipitates and clog the tubing. Once the tubing is clogged, the analysis can no longer be continued, and it therefore must be done over again after a costly and troublesome job like exchanging and washing the tubing. This is an unsolved major problem with the post-column fluorometric determination-boric acid complex anion exchange method.
Further, the sugars which can be analyzed using the post-column fluorometric determination-boric acid complex anion exchange method include monosaccharides such as glucose, mannose, galactose, fructose, and rhamnose; oligosaccharides such as maltose and maltotriose; amino sugars such as glucosamine, galactosamine; and uronic acid such as glucuronic acid
An example of analytes of this method is sugar chains of glycoproteins. Those reducing sugars which form sugar chains of glycoproteins include neutral sugars such as mannose, galactose and fucose; amino sugars such as galactosamine. Sugar chains sometimes contain mannose-6-phosphate (M6P), in which mannose is modified with one molecule of phosphoric acid.
M6P contained in sugar chains of glycoproteins has an important function to promote uptake of those proteins when they are being taken up into cells, through its binding to a mannose-6-phosphate receptor on the cell membrane. Lysosomal enzymes, such as iduronate-2-sulfatase (I2S) localized in lysosomes, are known as proteins containing M6P in their sugar chains (Non-patent document 2). Some lysosomal enzymes are produced by recombinant technology and used as therapeutic drugs for patients who hereditarily lack those enzymes. They are, for example, α-galactosidase A and glucocerebrosidase. Because lysosomal enzymes are enzymes which function within the cells, for those lysosomal enzymes administered to a patient in some manner, such as by intravenous injection, to exhibit pharmacological effects, they must be taken up into the cells of the patient, and for this to happen, it is necessary that the sugar chains of the lysosomal enzymes contain M6P. Thus, it is particularly important to analyze M6P when sugars forming the sugar chains of such enzymes are to be analyzed. While the post-column fluorometric determination-boric acid complex anion exchange method can be used as an analytical technique for a variety of sugars, no technique has been established to analyze M6P using this method.